Biochem. J. (2014) 462, 15–24 (Printed in Great Britain)

15

doi:10.1042/BJ20140295

REVIEW ARTICLE

*Department of Genetics, Development and Cell Biology, Crop Bioengineering Consortium, Iowa State University, Ames, IA 5001 U.S.A.

Genome editing is the practice of making predetermined and precise changes to a genome by controlling the location of DNA DSBs (double-strand breaks) and manipulating the cell’s repair mechanisms. This technology results from harnessing natural processes that have taken decades and multiple lines of inquiry to understand. Through many false starts and iterative technology advances, the goal of genome editing is just now falling under the control of human hands as a routine and broadly applicable method. The present review attempts to define the technique and capture the discovery process while following its evolution from meganucleases and zinc finger nucleases to the current

state of the art: TALEN (transcription-activator-like effector nuclease) technology. We also discuss factors that influence success, technical challenges and future prospects of this quickly evolving area of study and application.

INTRODUCTION

The ability to manage and exploit this powerful process would bring direct benefits to scientific, environmental, medical, agricultural and industrial arenas. However, until relatively recently, site-specific recombination could be routinely controlled in only a selected few species. The advent of high-throughput DNA sequencing and the enormous amount of data that are available from many ‘omics’ programmes underscore the need for a precise method to target and modify specific DNA bases within the genome of a broad range of organisms: we refer to this practice as genome editing. The present review will discuss the history, challenges, solutions and future of nucleasemediated genome editing with an emphasis on TALEN [TALE (transcription-activator-like effector) nuclease] technology.

Recombination is a mechanism by which cells repair damaged DNA and exchange information between identical or similar DNA molecules. This process is involved in many stages of the cell cycle and reproduction. It also aids in replication of some viruses, mobile elements and plasmids and plays a part in duplications, deletions and rearrangements of genetic material. In short, recombination appears to be essential for all known forms of life by acting both to ensure integrity of the genome and to contribute the genomic diversity needed for the action of evolutionary forces. Recombination is a broad topic and for purposes of this discussion we will limit this review to DNA repair events that occur following a DSB (double-strand break) and narrowly define these events as NHEJ (non-homologous end-joining) and HR (homologous recombination). NHEJ occurs when the ends of a DSB are re-joined and does not involve a DNA template to guide this repair. NHEJ can have many outcomes, such as restoration of the original sequence (a precise repair), or a disruption can occur by insertion or deletion of nucleotides or a combination of the two (an error-prone repair) (Figure 1A). Additionally, large deletions, inversions or translocations can occur (gross rearrangements) [1]. On the other hand, HR utilizes extended regions of homology between the exposed ends of a DSB and a donor DNA molecule, which is used as a template for repair [2]. Following a DSB, resection of the exposed 5 ends occurs resulting in free 3 singlestranded DNA that may invade a donor DNA. A free 3 end can act as a primer, and information may be copied from the donor DNA until polymerization ends, homology between the broken ends is found, gaps are filled in and breaks are sealed. Unlike NHEJ, HR can replace missing information at the DSB site; however, if the donor DNA template contains alternative information, then a gene conversion event may take place (Figure 1B).

Key words: genetic engineering, genome editing, homologous recombination (HR), meganuclease, NHEJ (non-homologous end-joining), transcription-activator-like effector nuclease (TALEN), zinc finger nuclease.

HISTORICAL PERSPECTIVE

The study of mobile intron replication and the subsequent characterization of their meganucleases led to experiments designed to control recombination, through targeted DSBs, in mammalian, amphibian and plant cells during the 1990s (Figure 2) [3–5]. These studies were the first to demonstrate that the long recognition sites of meganucleases could be used to induce sitespecific DSBs that could then be used to control recombination. These and other studies led to the engineering of meganucleases for use as genome editing tools [6–9]. Another form of engineered nuclease came with the discovery and engineering of the ZF (zinc finger) DNA-binding domain. A single ZF binds three consecutive bp of DNA and can have a cross-strand interaction with a fourth base. Engineered ZF modules were linked to form arrays that recognize 9, 12 or 18 consecutive bases and eventually led to ZF transcription factors and ZFNs (ZF nucleases) when fused to the non-specific nuclease domain of the restriction endonuclease FokI (Figure 3) [10].

Abbreviations: AD, activation domain; DSB, double-strand break; FLASH, fast ligation-based automatable solid-phase high-throughput; HR, homologous recombination; NHEJ, non-homologous end-joining; NLS, nuclear localization signal; RVD, repeat variable di-residue; TALE, transcription-activator-like effector; TALEN, TALE nuclease; Trex2, exonuclease 2; ZF, zinc finger; ZFN, ZF nuclease. 1 These authors contributed equally to this work. 2 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).  c The Authors Journal compilation  c 2014 Biochemical Society

Biochemical Journal

David A. WRIGHT*1 , Ting LI*1 , Bing YANG*2 and Martin H. SPALDING*2

www.biochemj.org

TALEN-mediated genome editing: prospects and perspectives

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Figure 1

D.A. Wright and others

Simplified models for the NHEJ and HR processes

Double-stranded chromosomal DNA is shown as two solid blue bars, the green box is the DSB point, the lightning symbol is an external DNA damaging agent, the checkered box is inserted DNA and the black double lines are donor DNA carrying a black box to represent change for HR. (A) Step 1, DNA is damaged resulting in a DSB as shown in step 2. In step 3, DNA is either repaired precisely or DNA is removed by repair enzymes and religated resulting in a deletion, as shown by the loss of the green box, or DNA is inserted, as depicted by the addition of the checkered box. (B) Step 1, DNA is damaged resulting in a DSB as shown in step 2. In step 3, DNA is removed by repair enzymes, then a free 3 end of the damaged DNA invades the donor DNA at a point of homology and repair enzymes copy information from the donor using the free 3 end of the damaged DNA as a primer. After extension, homology is found between the free 3 ends of the damaged DNA and the break is repaired by further polymerization and ligation as in step 4, resulting in gene conversion in the chromosome as symbolized by the black box.

Initial engineered ZFN experiments demonstrated HR using an extrachromosomal target in Xenopus oocytes, NHEJ followed by HR at an endogenous locus in Drosophila, marker gene restoration in human cells, NHEJ at an artificial target in Arabidopsis and HR at an integrated marker gene in tobacco [11–16].  c The Authors Journal compilation  c 2014 Biochemical Society

After these initial successes, many model and economically important organisms were subjected to meganuclease and ZFNmediated genome editing with a notable record of success. However, implementation of either technology proved to be technically challenging. Problems with toxicity, off-target

TALEN-mediated genome editing

17

(nuclear localization signals) and a highly conserved acidic AD (activation domain) (Figure 4A) [19]. The central repeats are nearly identical except for two amino acids at positions 12 and 13, called RVDs (repeat variable di-residues) [20]. Different TALEs vary in repeat number and RVD composition [19]. It is their role as plant pathogenesis factors that initially captured the attention of researchers, but their simple DNA-binding code and ease of engineering has led to their widespread use as artificial transcription factors and nucleases. TALE DNA-recognition code

Figure 2 A simplified model for homing nuclease-mediated mobile intron replication In step 1, double blue bars indicate double-stranded DNA and the green box represents the target site of the intron-less allele. Black double bars represent the intron-containing allele and the long wavy box indicates the mobile intron encoding a nuclease. In step 2, the mobile intron expresses its nuclease, as depicted by the vertically striped red oval, which binds to the target site and cleaves the DNA as shown in step 3. In step 4, DNA is removed at the DSB site by repair enzymes, then a free 3 end invades the intron-containing allele at a point of homology, and repair enzymes copy the intron from the donor using the free 3 end of the damaged DNA as a primer. After extension, homology is found between the free 3 ends of the damaged DNA, and the break is repaired by further polymerization and ligation as in steps 4 and 5. The process results in replication of the mobile intron into the formerly intron-less allele, as shown by the conversion of the heavy bars into light bars.

cleavage, limited target choice and difficulties with nuclease engineering have plagued the system even as progress has been made to address these shortcomings. TALEN TECHNOLOGY

The TALEs represent the largest family of type III effector proteins from Xanthomonas spp., a group of Gram-negative bacterial plant pathogens, with TALE homologues also in Ralstonia solanacearum [17,18]. TALEs contain an N-terminal bacterial secretion and translocation signal, a single unique DNAbinding domain, termed repeat 0, followed by a central repeating modular DNA-binding domain, two or more C-terminal NLS

A number of clues led to the discovery of the TALE DNA-binding code. TALE AvrXa7 binds dA/dT oligonucleotides, TALEs with similar RVDs bind similar targets and TALE target sequence length is nearly equal to TALE RVD number [21–23]. In 2009, two independent groups using different methods reported the TALE DNA-binding code in the same issue of Science. The first group used available information to statistically surmise that each RVD displayed a preference for specific nucleotides and correlated this with public microarray infection data [20]. The second group used alignments to predict TALE targets followed by molecular biology methods to experimentally validate predictions [24]. The code, as it turns out, is relatively simple: the first base of the target is recognized by a region in the TALE N-terminus (repeat 0) and the remaining bases are sequentially bound by one RVD per nucleotide with one type of RVD preferentially recognizing one specific nucleotide. Many RVDs have been described in the literature; the four major RVDs are HD, NI, NG and NN, which predominantly bind to cytosine (C), adenine (A), thymine (T) and guanine (G) or adenine respectively. Each RVD has the capacity to recognize other bases, although the interactions indicated above are the most common. It should be noted that TALEs are more sensitive to target site mismatches at the 5 end relative to the 3 end. This is due in part to the polarity TALEs exhibit regarding target sites and the fact that repeat 0 is generally specific for thymine. Mutation of this initial thymine usually abolishes TALE binding activity, and other 5 mutations make a TALE less likely to bind, whereas 3 mutations are likely to have less of an effect, since the more C-terminal repeat regions show less affinity for DNA [25]. TALE structure

Shortly after discovery of the TALE DNA-binding code, the structure of the repeat domain was solved through protein crystallization by two groups [26,27]. Crystal structure analysis sought to determine how TALE repeats are ordered and how they interact with specific DNA bases. Examination of TALE dHax3 and TALE PthXo1 indicated that the central repeat region is a right-handed super helix wrapping around the sense strand of the target DNA, and each repeat is a lefthanded helix–loop–helix structure. The 12th and 13th amino acids (RVD) of the repeats are located in the loop region where the 12th amino acid stabilizes the loop through hydrogen bonds with the protein backbone, whereas the 13th amino acid determines base specificity when interacting with the major groove. Analysis of repeat 0, which precedes the central RVD repeats, revealed a degenerate helix–turn–helix motif. A tryptophan (Trp232 ) in the turn region specifically interacts with the conserved 5 thymine in the target [27]. These crystal structures provided great insight into the TALE DNA-binding domain architecture and will help guide future engineering efforts.  c The Authors Journal compilation  c 2014 Biochemical Society

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Figure 3

D.A. Wright and others

Depiction of a pair of ZFNs

Individual ZFs are shown as coloured ovals, marked F1, F2 and F3, and the FokI nuclease domain is indicated by the red striped oval marked N. Small N and C indicate termini. Black lines indicate binding of individual ZFs to DNA triplets, and dotted lines indicate cross-strand interactions. In step 1, individual ZFNs bind their target DNA to form a pair, with nucleases positioned over a six base spacer. In step 2, the nuclease domains form a dimer interface and cleave the target DNA, leaving a four base 5 overhang. This is followed by repair using a NHEJ or HR pathway, as shown in step 3. Note that ZFNs bind DNA backwards with respect to its N-terminus and the 5 DNA end.

Figure 4

A TALE activator and a pair of TALENs

Repeat 0 is shown as a green oval marked with R0 and individual RVDs are depicted as small coloured rectangles with their nucleotide-binding code indicated. The large yellow box in (A) marked AD, represents a transcription AD, and the large red striped ovals in (B), marked N, represent tethered FokI nuclease domains. Small N and C indicate termini. In (A), an engineered 18 RVD TALE transcription factor is illustrated, as indicated by the truncated N-terminus and added C-terminal AD. It recognizes a target that is 19 bp long and activates transcription near the target site. In (B), an engineered 18 RVD TALEN pair is illustrated that positions FokI nuclease domains over a 12 base spacer, where they will cleave the DNA to mediate NHEJ or HR. Note that the TALE DNA-recognition domain binds DNA in a forward orientation with respect to its N-terminus and the 5 DNA end.

TALENs

FokI homodimer and heterodimer forms

Like ZFNs, TALENs are generated by fusing the FokI nuclease element to a TALE DNA-binding domain and TALEN target half-sites are chosen such that pairs are arranged in an opposing orientation on opposite sides of double-stranded DNA with an optimal spacer sequence between them (Figure 4B). Results of side-by-side comparisons of ZFNs and TALENs suggest that TALENs exhibit reduced nuclease-associated cytotoxicity [28]. This may be due in part to longer DNA target sites and the thymine requirement at the 5 end of a target site, together resulting in greater specificity, but much more comparative research is needed before more definitive conclusions can be drawn.

Despite its nearly universal adoption for artificial nuclease construction, the FokI nuclease domain has some drawbacks: most notably is that it normally functions as a homodimer. When two identical FokI nucleases are brought into proximity by their DNA-binding domains, they dimerize and cleave the DNA target. The disadvantages of the homodimer become apparent when it is understood that artificial nuclease pairs have left and right halves. When these halves are designed with a homodimer FokI, they can have three interactions: two left halves or two right halves can make a functional nuclease just as easily as the intended interaction between the left and right halves of a nuclease pair. In

 c The Authors Journal compilation  c 2014 Biochemical Society

TALEN-mediated genome editing Table 1

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Variant DNA cleavage domains of FokI with improved specificity and activity

Name

Mutations

Notes

Reference

RV-DA KK-EL ELD-KKR

Left D483R and I538V and right R487D and I499A Left Q486E and I499L and right E490K and I538K Left Q486E, I499L and N496D and right E90K, I538K and H537R

Nuclease activity compromised in vitro and in vivo

[33] [31] [29]

Sharkey

S418P and K441E

RE

S418R and K441E

Table 2

Higher nuclease activity compared with other heterodimers and increased temperature tolerance Improved nuclease activity and compatible with heterodimer forms Increased DNA binding and decreased nuclease activity. Can be incorporated into a heterodimer format

[30] [32]

Different architectures of TALE and FokI fusion

N/A, not applicable. N-/C-terminal truncation

Spacer requirement (bp)

FokI type

Codon bias

Reference

288/295 (full-length) 152/63 287/231 153/17, 153/47 111/42 152/14 152/18 240/63 Modified 152/63

16–31 12–21 15–27 12, 12–15 N/A 12–14 13–16 14–16 15–16

Homodimer Homodimer Homodimer KV/EA N/A DAS/RR Sharkey Homodimer NEL/CKK Homodimer

Native TALE Native TALE Native TALE Native TALE Native TALE Human codon Native TALE Silkworm codon Native TALE

[37] [43] [44] [28] [25] [36] [45] [46] [47]

essence, using the homodimer is like simultaneously introducing three different nucleases into a cell. Compounding the problem is the possibility of a TALEN binding to sites that are similar in composition to the intended target DNA. As a result, there may be TALEN molecules bound to many locations in the genome and in multiple combinations. The probability of overwhelming a cell with DSBs becomes high, precipitating cell death and collateral damage in the genome of surviving cells. To reduce off-target toxicity, several obligate heterodimer versions of FokI have been developed. The engineered versions are based on structure-guided design, DNA shuffling and mutagenesis [29–33]. These designs have greatly reduced the toxic effect of off-target DSBs by limiting the pairing of TALENs to just the intended left and right halves. The trade-off is that many heterodimer FokI designs have lower nuclease activity, which may reduce the number of expected recombination events. Example heterodimer pairs are summarized in Table 1. TALEN architectures and assembly methods

The TALE family of engineered nucleases is diverse: the protein itself is relatively large, there is a host codon bias and the FokI attachment sites are numerous. These variables lead to a wide range of engineered TALE architectures in the literature. For example, FokI can be directly fused to the existing TALE structure or a TALEN can be generated with a combination of N-terminal and/or C-terminal deletions. C-terminal deletions affect the spacer length requirement (typically 12–31 bp) between half-sites and may or may not remove the NLS and ADs. Nterminal deletions generally remove the bacterial secretion and translocation signals and may require the addition of an NLS. TALEN RVD assembly methods are similarly varied and include PCR, golden gate, FLASH (fast ligation-based automatable solidphase high-throughput), LIC (ligation-independent cloning),

iterative capped assembly and commercial services [34–42]. Some of the golden gate methods and FLASH are reported to be suitable for high-throughput automated TALEN production. With so many combinations, there are many open questions relating to TALEN architecture and assembly, which will remain largely unanswered until rigorous comparative studies between architectures are carried out. Table 2 lists some of the more popular architectures and assembly methods.

TALEN application

Quick and easy assembly, availability of powerful resources, cross-species flexibility plus a high rate of success, together make TALEN technology an easy choice for genome editing applications. In fact, more reports of TALEN usage have been published in the last few years than for all previous artificialnuclease-based technology applications from the last few decades. TALEN technology has been used in a wide range of organisms, including yeast, plants, algae, protozoan, nematodes, fish, insects, mammals and human cells. The short time since the initial TALEN reports to the plethora of species applications demonstrates the simplicity and general applicability of TALEN technology.

Application in animals

Early reports of TALEN application in animal cells showed NHEJ and HR events in human cells (9 %–21 % and 16 % respectively), NHEJ in the progeny of Caenorhabditis elegans (3.5 %), and NHEJ in zebrafish (up to 12.5 %) [43,48,49]. A recent demonstration includes TALEN-mediated disruption of the mouse Y chromosome [50]. In a broader TALEN study, using Bos taurus (cow) and Sus scrofa (pig), it was demonstrated that 23 of 36 TALEN pairs had high activity in primary cells at 15  c The Authors Journal compilation  c 2014 Biochemical Society

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D.A. Wright and others

loci resulting in mono- and bi-allelic knockouts. In addition, up to 75 % of TALEN-injected embryos resulted in knockout events [51]. These reports clearly demonstrate efficient TALENmediated genome editing for animal cell application.

Application in plants

TALEN technology has been used in dicot species such as tobacco and Arabidopsis and in monocot species such as rice, Brachypodium, barley and maize [52–56]. TALEN-mediated genome editing in plants was first reported in rice, where the OsSWEET14 (Os11N3) promoter region was disrupted through NHEJ, which resulted in plants that were resistant to Xanthomonas oryzae [57]. In another example, the phytic acid biosynthesis pathway was targeted in maize, which produced independent transformants at four targeted loci with NHEJ-based disruptions in approximately 39.1 % of the transformants [53]. These studies indicate that TALEN-mediated genome editing technology can precisely modify predetermined loci in crop plants leading to improved varieties that are more environmentally friendly and disease or stress resistant.

Understanding gene function

The simplicity and speed of TALEN production easily lends itself to identifying the role of thousands of genes whose functions remain unknown. For example, when knocked out, the nuclear gene NDUFA9 (NADH dehydrogenase 1 α subcomplex 9 39 kDa) was identified as an assembly factor that stabilizes the junction between the membrane and the matrix arm in the mitochondrial respiratory chain complex I [58]. In 2013, three independent groups successfully targeted and disrupted microRNAs with TALENs in a mouse model and in human cells, leading to the construction of a human cell microRNAdeletion library containing 274 loci [59–61]. Additionally, by placing TALEN pairs under the control of a heat-shock promoter or a tissue-specific promoter, mutations were generated at precise developmental stages or in specific somatic tissues in C. elegans and Ciona [62,63].

Large-scale genome editing

When two pairs of TALENs are used to simultaneously target different loci in a cell, this action can result in a large deletion, inversion or translocation. This strategy has many benefits when studying gene clusters, microRNAs, long non-coding RNAs that do not respond to frame-shift mutations or the effects of gross genome rearrangements. Studies have demonstrated that the size of deletion can range from 795 bp to 5.5 Mb when a dual TALEN strategy is applied [46,64]. In addition to deletions, inversion events also have been reported [51,54,64]. For example, when two pairs of TALENs were simultaneously injected into pig fibroblast cells, large deletions and inversions were detected in 10 % and 4 % of injected cells respectively [51]. Another intense area of interest is the study of chromosomal rearrangement in cancer cell biology. For example, modelling gene rearrangement through TALEN technology contributed to the discovery of a drug-resistance mechanism in prostate cancer [65]. In another report, cancer cells were generated de novo, and a pre-existing translocation in a cancer cell line was reversed using TALENs to restore the native chromosome arrangement [66].  c The Authors Journal compilation  c 2014 Biochemical Society

FACTORS THAT AFFECT TALEN EFFICIENCY

TALENs have been successfully used in more than 25 species, resulting in hundreds of publications. This broad application complicates any general evaluation of TALEN efficiency, because of a variety of factors, such as the species involved, the donor DNA for HR work, type and amount of molecule delivered, delivery methods, TALEN architecture, target choice, and detection methods. A good assessment of TALEN effectiveness will depend on a thorough evaluation of the literature that is most relevant to the species of interest. With that said, some general information gleaned from the literature can help with experimental design.

Donor DNA for HR

Relatively few thorough HR experiments have been reported, but it is clear that some species engage HR more readily than others. Important considerations for HR experiments are the length of the donor DNA molecule, the position of the desired change relative to the DSB site, the level of heterology, inclusion of the nuclease target site in the donor and the size of inserted DNA, if any, relative to the target. Reported donor DNAs range from about 4 kb down to oligonucleotides and can be single or double stranded. The window of incorporation of genetic changes can be narrow, as demonstrated with I-Sce I in mouse cells, where 80 % of events having track lengths of 58 bp or less for HR occurred on either side of the DSB [67]. Although less stringently tested, a similarly narrow HR window was observed in two independent HR experiments using human cells [68,69]. In plants the story may be different. Although the HR frequency was reduced from 4 % at a ∼160 bp to 0.2 % at a ∼1500 bp distance from the DSB, the HR window in tobacco is relatively wide [70]. Donor DNA divergence is another area that requires more study, although it has been shown that as little as 1.2 % heterology can reduce HR by approximately 6-fold in mammalian cells [67]. Additionally, removing the nuclease-recognition site from the donor DNA to prevent recutting of the HR event had surprisingly little effect on HR efficiency [69–71]. Furthermore, several studies have shown relatively high incorporation rates for large DNA segments into DSB sites using plant and animal cells, which suggests insertions may be treated differently than donor DNA-containing nucleotide substitutions [16,69,72]. Until more information is available, longer donor DNAs with a short relative distance between the DSB and the desired DNA change and with low heterology may be good choices for donor DNA design.

Delivery molecule

If DNA is the delivery vehicle for TALENs, there are a number of structural parameters to consider, along with the fact that DNA will leave behind a trace in the genome. Important considerations should include promoter strength and tissue specificity, independent expression or coupled expression using, e.g. an FMDV 2A type linker [56,57], TALEN architecture and codon bias, to name a few. If TALEN DNA in the genome is undesirable, then the sexual nature of the cell is also a consideration. For species that can be genetically crossed, traces of the transgenic TALEN DNA can be removed genetically, but there is a cost both of time and money associated with breeding programmes. For species that cannot be bred, the use of RNA or protein may be a better choice to avoid retention of the transgenic TALEN DNA in the final product.

TALEN-mediated genome editing

RNA is a viable nuclease delivery form for some species, and it has the benefit of not leaving exogenous DNA in the cell [51,73]. However, use of RNA adds to the complexity of genome editing, because it is much more susceptible to degradation than DNA. Additionally, the generation of RNA is more cumbersome than DNA. The RNA needs to be of high quality, produced in large quantities and have a 5 cap and a poly(A) tail to be effective. Additionally, a growing body of evidence suggests that there may be less toxicity associated with nucleases when exposure time is limited; use of RNA is one way of limiting that exposure time [14,74]. The protein form of a nuclease can be taken up directly by some cell types if a cell-penetrating peptide is present or, as demonstrated with ZFNs, the protein naturally passes through membranes [75]. Examples of cell-penetrating peptides include the R9 (polyarginine) and TAT (from HIV) motifs. It was recently reported that TALENs bearing either cell-penetrating peptide can be taken up by human cells to efficiently mediate NHEJ. A TALEN conjugated to the R9 motif reportedly led to gene disruption in 15 % of HeLa cells, whereas a TALEN fused to TAT led to a knockout in 5 % of human-induced pluripotent stem cells [76,77]. Coupling TALENs and cell-penetrating peptides is a relatively new area of exploration and more research is needed before general conclusions can be drawn.

Delivery method

TALEN delivery is largely dictated by the species and tissue type, but some general observations can be made. For animals there is a wider range of delivery methods available, and transfection efficiency is generally high, whereas plants have fewer options, and the relative transformation efficiency is generally lower. Nuclease delivery for animal cells includes such methods as nucleofection with RNA or DNA, lipofection, microinjection of either RNA or DNA, particle-based methods, and virusmediated transfection. Differences in concentration and form of the delivery molecule, either RNA or DNA, can contribute to genome editing efficiency. For example, it has been demonstrated that modification frequencies in rat embryos microinjected at the single cell stage with DNA are lower than with RNA, and that observed frequencies may be dose dependent [78]. For viral delivery, it has been demonstrated that adenovirus can deliver TALEN genes into various human cell types, but rearrangements of TALENs was a problem when performing lentiviral vectormediated transfection [79]. For plants, the higher efficiency delivery methods reported fall into three basic areas: electroporation, physical methods such a biolistics and Agrobacterium tumefaciens infection. Electroporation most commonly involves DNA and is usually limited to the few species that can be regenerated from protoplasts. Biolistics and A. tumefaciens infection are more widely applicable and normally involve DNA, either as plasmids or T-DNA respectively. However, compared with animal cells, both methods are relatively inefficient [80]. A drawback of A. tumefaciens delivery is that transformation of a single cell by two different A. tumefaciens, each harbouring one component of a TALEN pair, is difficult to achieve. Owing to this constraint, both TALENs in a pair are normally encoded by one plasmid, either expressing the two TALENs separately or by using a single promoter and linking both TALENs by an FMDV 2A ribosomal skipping peptide [56,57]. A very recent paper demonstrated the delivery of nucleases to tobacco cells using a modified Geminivirus system that increased genome editing by one to two orders of magnitude over A. tumefaciens-based delivery methods [81].

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Target choice

To a certain degree, DNA composition, half-site recognition lengths and spacer length can affect TALEN performance. Halfsites commonly range from 12 to 24 RVDs, and spacer length is determined by length and structure of the C-terminus and FokI fusion point, with a shorter C-terminus generally decreasing the optimal spacer length. Base composition of the target should also be carefully considered. Some rules have been suggested in the literature, but these have not been rigorously tested. Beginning the target half-site with a thymine, use of longer half-sites with a good mix of bases and considering spacer length requirements are good starting points for target selection. The type of intended recombination event is also a factor. If gene disruption by NHEJ is desired, the target should be closer to the 5 end of the gene’s coding sequence and placed in an exon or on an intron–exon border. If HR is the preferred outcome, the target site should be as close to the point of change relative to the donor as possible.

Mutation detection

For mutation detection, one of the most commonly used methods is a DNA mismatch cleavage assay. For this assay, wildtype and putative mutant PCR products are annealed, then a mismatch-sensitive endonuclease, such as Cel1 (Surveyor) or T7 endonuclease I, digestion is performed. Estimates of nuclease activity are made using gel densitometry to detect cleavage at the site of mismatches [31,82,83]. Another reported detection method relies on RFLP (restriction fragment length polymorphism) analysis, but requires an appropriate restriction site in the target site spacer region to accurately estimate event frequency [49]. This method can be enhanced through pre-digestion of genomic DNA with the respective restriction enzyme before PCR amplification. More sophisticated mutation detection methods have also been reported that use PCR product mismatches as a basis for an assay. For example, HRMA (high-resolution melt analysis) takes advantage of the thermostability difference between perfectly matched and mismatched PCR fragments [59,84]. Additionally, the HMA (heteroduplex mobility assay) uses PAGE or microchip electrophoresis for easy high-throughput mutation screening. This assay relies on the observation that heteroduplexes have reduced gel mobility proportional to their degree of divergence [85]. A similar strategy is described for screening transmission efficiency by observing PAGE migration of relatively small PCR fragments and searching for altered fragment sizes [86].

NHEJ exonuclease enhancement

As effective as TALENs are at inducing mutations through NHEJ, methods have been reported to improve their efficiency. Enhancing the genome editing potential of DSBs by decreasing the probability of precise repair is one way to improve overall TALEN efficiency. A recent example of this is the coupling of TALENs with an exonuclease to remove nucleotides from the DNA ends at the DSB before a precise repair can be made. Coupling TALENs with Exo1 (exonuclease 1) increased mutagenesis efficiency up to 30 % in rat cells [87], whereas coupling to Trex2 (exonuclease 2) increased efficiency approximately 144 % with no obvious toxicity in human cells [88]. A paper describing a meganuclease TALE fusion also reports increased efficiency when coupled with Trex2 [89].  c The Authors Journal compilation  c 2014 Biochemical Society

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D.A. Wright and others

Organism

Repression domain

Reference

as newer genome-modifying technologies such as CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) are developed.

Arabidopsis Mammalian cells E. coli Drosophila

EAR-repression domain (SRDX) mSin-interaction domain (SID) No repression domain; transcription simply physically blocked Kr¨uppel and Hairy repression domains; Hairy reportedly stronger

[97] [95] [98] [96]

FUNDING

Table 3

Activators or suppressors derived from TALEs

ENGINEERED TALE TRANSCRIPTION FACTORS

As discussed above, the native function of a TALE is to act as a eukaryotic transcription activator after being produced by a bacterial pathogen and secreted into a host cell. TALEs contain an NLS and an acid AD, but their basic transcriptional mechanism has not been clearly demonstrated. In plants, a native TALE shifts the transcription start site of a target gene from its normal location to a position 44–61 bp downstream of the TALE target site, or in the case of some TALE-activated plant defence genes, the transcription start site can be basically unchanged [19,90]. Even with limited information, it is easy to speculate that engineered TALEs can be used as specific transcription factors for activation and repression of target genes. When designing engineered TALE transcription factors, the position of the target sequence appears to be relatively flexible. Positions as far as 500 bp upstream of the native target gene transcription start site have reportedly been successful, as have positions upstream, downstream or overlapping the TATA box [91]. Additionally, multiple TALEs targeted to different regions of the same promoter appear to act synergistically [91,92]. This may be due to the creation of additional open chromatin as a result of TALE binding, but contradictory results cloud this issue, and additional evidence is needed to determine whether TALEs bind better to open or compact chromatin and whether they can open silent chromatin [91–93]. Additional modifications of engineered TALEs also have reportedly improved their functions as activators. For example, it has been demonstrated that the native acidic AD can be replaced with the herpes virus VP16 AD [94]. In mammalian cells, TALEs usually display higher activity when using VP16 or VP64 fused with a commonly used TALE truncation version, 152/ + 95 or when used with the FLASH system [43,91]. It has also been demonstrated that the TALE acidic AD can be replaced by a repressor, such as the SID (mSin-interaction domain) and that this fusion can reduce expression of a target gene in plant and animal cells. Additionally, it has been reported that just physical binding of a TALE can block transcription in bacteria [95–98] (Table 3). CONCLUSIONS

The development of TALENs and related TALE technology is moving at a fast pace. The user friendly structure and availability of public resources have already changed the perception of DNAbinding domain engineering from one of difficulty to one of ease, which has appealed to a broader audience of researchers. In response, volumes of data have been published demonstrating TALEN-mediated genome editing and the utility of other TALEbased technologies in a wide variety of organisms. The technology still presents some challenges, and there are many unknowns. Over time, solutions will be presented and many more discoveries will be made. TALE technology appears to be positioned to move targeted manipulation of the genome well beyond previous platforms and will be useful into the foreseeable future even  c The Authors Journal compilation  c 2014 Biochemical Society

The authors thank the U.S. National Science Foundation [grant numbers 1238189 (to B.Y.) and MCB-0952323 (to M.H.S)] and the U.S. Department’s Advanced Research Projects Agency-Energy Program [grant number DEAR0000010 (to M.H.S.)] and Office of Science, Basic Energy Science Division [grant number DE-FG02-12ER16335 (to M.H.S.)] for support of the research programmes in the two laboratories.

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 c The Authors Journal compilation  c 2014 Biochemical Society

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